Physical, chemical, and biological cues within stem cell microenvironments are known to influence differentiation in vivo (1). To understand how these cues impact stem cell fate, researchers have searched for characteristics such as conditions, size, and temperature that facilitate optimal development through variations in extracellular matrices, culture conditions, and temporary exposure to chemical growth factors (2-6, 11). Yet, even with currently available microtechnology, our understanding of how a cell’s physical shape and geometry influence differentiation has been slow to develop. Now, researchers at the University of Chicago have taken advantage of a microcontact printing technique to culture human mesenchymal stem cells (MSCs) in a variety of shapes and patterns, to control differentation into osteogenic or adipogenic cell lineages.
In 1997, Ingber, Whitesides, and colleagues demonstrated the use of geometrically patterned substrates to control the microenvironments of individual cells and control cellular apoptosis (7). More recently, Chen and colleagues demonstrated cell shape and size can direct MSC fate (8). While these studies helped establish the roles of adhesion and cytoskeletal tension in influencing stem cell fate, the mechanical process that controls these traits was unknown. But the University of Chicago research team, led by chemistry professor Milan Mrksich, hypothesized that changes in MSC culture shape would regulate variations in adhesion and cytoskeletal tension and consequently influence MSCs differentiation.
“Previously we had found that the local geometric features of shapes can have a strong influence in directing the positions of cytoskeleton elements in the cell,” Mrksich told BioTechniques. “Specifically, shapes that have 'pointy' features are effective at promoting strong focal adhesion contacts to the substrate and in turn support a contractile cytoskeleton.” Based on the work of Chen and Dennis Discher of the University of Pennsylvania (9-10), Mrksich and his team expected cells with a tense cytoskeleton to become bone cells. In contrast, shapes with broad sweeping edges characteristically result in cytoskeletons that are less contractile and therefore should generate adipose cells. “These shapes were selected as a way of tuning the contractility of the acto-myosin cytoskeleton,” said Mrksich.
Mrksich’s team utilized microcontact printing, a technique that uses a relief pattern on a polydimethylsiloxane (PDMS) stamp to form patterns of self-assembled cell monolayers on the surface of a substrate through conformal contact. They created their PDMS stamps as a pattern for adhesive islands of the organic chemical octadecanethiolate on a glass cover slip, coated with a layer of gold. When octadecanethiolate [its chemical composition is CH3(CH2)17SH] reacts with gold (Au), bonds form between CH3-S and Au-S. These bonds are strong in the favor of CH3-S, which makes it easier for the chemical to pull electrons from the surface of the gold. Certain regions of the culture were modified with a tri(ethylene glycol)–terminated monolayer and immersed in a solution containing fibronectin. Finally, MSCs were layered onto these regions of the substrate.
“There are dozens of patterning techniques that can be adopted, but microcontact printing is probably the most reliable, simplest to use, and applicable to the patterning of large area substrates,” said Mrksich.
Three culture shapes were tested: a flower, a pentagon, and a star. The flower shape had large convex curves along the edges, and this culture yielded 62% adipogenic cells, with the remaining cells forming osteoblasts. The pentagon, with straight edges, showed a relatively even split between forming adipogenic and osteogenic cells. The researchers then tested a star, with concave edges and sharp points at the vertices. This culture resulted in 62% osteogenic cells with the remaining cells differentiating into adipogenic cells. Because the shapes all had a consistent surface area, and the medium and soluble factors were unchanged, the researchers say shape cues imposed by the underlying patterns were responsible for the observed changes in cell fate.
Shape is able to influence cell lineage differentiation in this way because cells assemble stress fibers along edges that overlap regions of substrate that are nonadhesive. For example, in the flower shape, stress fibers formed at the acute corners between “petals” where the regions were nonadhesive. The star shape sharply contrasted with this by developing highly contractile regions in the concave regions between the points of the star across the nonadhesive area. According to the researchers, this shows that cytoskeleton in adherent cells is strongly influenced by subtle geometric shape cues, suggesting that osteogenic cells are directly dependent on a contractile cytoskeleton.
Mrksich says more work is needed for this culturing technique to have an impact on the field of human stem cell therapy. “I don't think we are ready yet to create patterned materials for the production of stem cells,” said Mrksich. “But the work does show the benefits of using patterned surfaces to remove the heterogeneity that is normally intrinsic to cell cultures and further, to understand the relationships between cell shape and function. I expect that this approach will find broader use in the study of signaling pathways and ultimately provide us with a comprehensive understanding of the functional consequences of cell shape.”
Mrksich’s laboratory is continuing to explore the influence of shape on cell culture. They are currently working to create surfaces where the stiffness of the substrate and presentation of extracellular matrix ligands can be modified along with shape. “This work is bringing further definition to the cellular microenvironment and will be important in understanding how these distinct factors combine to influence cell fate,” he said.
Funding for this research was provided by the National Cancer Institute and the National Institutes of Health. The paper, “Geometric cues for directing the differentiation of mesenchymal stem cells,” was published March 24 in Proceedings of the National Academy of Sciences.
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